| The phylogeny of the complement system during evolution has become one of the key problems in immunology. The studies on complement have been over 100 years, but it remains unclear when and which animal is initially equipped with the ancestral complement system. Here, we report the results of the study on the complement system of the cephalochordate amphioxus and lower vertebrates lip shark and grass carp. This can help us not only betters our understanding of the phylogeny of the complement system during chordate evolution but also provides an important link between innate and adaptive immune responses.Amphioxus or lancelet, a cephalochordate, is the extent invertebrate most closely related to the vertebrates. We have invented a technique to extract the humoral fluid from the amphioxus Branchiostoma belcheri tsingtauense. The presence of complement-like activity in the humoral fluid of amphioxus was examined. The complement classical pathway (CP) activity is assayed by determining the amount of serum required to hemolyse a given number of sensitized sheep red blood cells (SRBC), whereas the complement alternative pathway (AP) activity measured in the same manner, but by using rabbit red blood cells (RaRBC) instead of sensitized SRBC. It was demonstrated that functionally and structurally similar complement components are present in the humoral fluid of amphioxus. The humoral fluid fromamphioxus was capable of causing hemolysis of rabbit erythrocytes and those obtained from species representing mammals, birds, amphibians and fish, but not sensitized sheep erythrocytes. There was no relationship between phylogenetic proximity of erythrocyte species to Branchiostoma and degree of hemolysis. The hemolytic activity was optimally assayed at 20癈, at pH 7.5, and in the presence of 10 mM Mg2+. The hemolytic activity was Mg2+-dependent and heat-sensitive, and was abrogated by treatment with rabbit anti-human C3 serum, zymosan, methylamine, hydrazine, and phenylmethylenesulfonyl fluoride (PMSF). In addition, titration by turbidimetric immunoassay (TIA) using sheep anti-human C3 serum revealed that amphioxus humoral fluid contained C3-like component, and its concentration is about 1.17 mg/ml, which is comparable to C3 concentration in human or dog sera. These suggest that the hemolytic activities displayed by amphioxus humoral fluid appear to represent the vertebrate complement system probably operating via the alternative pathway (AP).The presence of lectins in the humoral fluids of amphioxus Branchiostoma belcheri tsingtauense, was also studied. It was showed for the first time that lectins were present in the humoral fluids of amphioxus. The hemagglutinating activity of the fluids was not affected by treatment with 50 mM EDTA and EGTA, while it was increased by treatment with 3 -ME. Therefore, hemagglutination activity of amphioxus humoral fluids is Ca2+-independent, but SH group-dependent. Hemagglutinating activity of the humoral fluids was inhibited by methyl- β -D-galactopyranoside, thiodigalactoside, D-galactose and asialofetuin. In contrast,N-acetyl-D-galactosamine, sucrose, D-glucose, glycogen, D-mannose, L-fucose and fetuin had no inhibitory effects on hemagglutination. So the lectin activity in the humoral fluids of amphioxus is SH group-dependent and is specifically inhibited by various D-galactosides. Also, the humoral fluids obtained from E. coli-injected amphioxus showed increased agglutinating activity against human B and O, rabbit, grass carp and toad erythrocytes, but not against human A and chick erythrocytes, hinting that there might be two types of lectins in amphioxus humoral fluids.To compare the complement lytic system between cephalochordate and cartilaginous fish, lip shark (Chiloscyllium plagiosum) serum complement activities were investigated. In this part, we reported for the first time the hemolytic activities of lip shark serum via the complement alternative pathway (AP). Lip shark serum was capable of hemolyzing rabbit erythrocytes and those of the vertebrate spe...
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